Low molecular weight thiol-containing compounds play a key role in protecting cells from the toxic effects of ionizing radiation, other free-radical-generating reactions, reactive oxygen species and chemical toxins. The only thiol-containing amino acid, cysteine, is considered to be a relatively minor cellular component in its free form, often reported to be present at concentrations of only a few micromolar in normal tissues (Loh et al., 1990, Rad. Res. 121:98-106). The presence of cysteine in the tripeptide glutathione (GSH) provides a much greater reserve of intracellular thiols than does free cysteine. Total intracellular concentrations of glutathione are typically more than 1 mM and have been reported to be as high as 25 mM in some cells (Post et al., 1983, Biochem. Biophys. Res. Comm. 114:737-742). Intracellular protein thiols comprise an additional 25 mM cysteine, while extracellular proteins, or the extracellular portion of transmembrane proteins, contain predominantly cysteine-cysteine disulfides (cystine) (Jocelyn, 1972, Biochemistry of the sulfhydryl group, Academic Press, New York). Enzymes such as glutathione-S-transferase and glutathione peroxidase have evolved to use glutathione, not cysteine, as the nucleophilic acceptor or reductant in cellular protection against toxic xenobiotics (particularly electrophiles) (Reed, 1990, Ann. Rev. Pharm. Toxicol. 30:603-663; Fahey and Sundquist, 1991, Adv. Enzym. 64:1-53).
Studies on the contrasting roles of radiation sensitization by oxygen versus protection by aminothiols have, in similar fashion, emphasized the role of glutathione. Despite this emphasis on the role of glutathione, it remains important to consider the auxiliary role of free cysteine, particularly in protecting cells from damage produced by ionizing radiation. This is because cysteine, due to its neutral charge and smaller size, is expected to have greater access to the negatively charged DNA and, therefore, is expected to provide greater protection from the damaging effects of ionizing radiation. (Zheng et al., 1988, Rad. Res. 114:11-27; Bump et al., 1992, Rad. Res. 132:94-104). However, the prior art does not disclose that tumor cells might have increased concentrations of free cysteine or that this might be a possible mechanism of radiation resistance in tumor cells.
Studies concerning the role of free cysteine in protecting cells from ionizing radiation have been hampered by the fact that cysteine is inherently difficult to measure except as part of the total non-protein sulfhydryl (NPSH) pool (Ellman, 1959, Arch. Biochem. Biophys. 82:70-77). For instance, there are no sensitive biochemical assays for the measurement of free cysteine analogous to the recycling assay for measuring glutathione levels developed by Tietze (1969, Anal. Biochem. 27:502-522). Further, development of more specific assays, based on HPLC, has been hindered by the fact that cysteine and its derivatives are extremely hydrophillic. Thus, cysteine and its derivatives tend to elute with the solvent and salt fronts used in HPLC rendering them difficult to resolve using this method (Allison and Shoup, 1983, Anal. Chem. 55:8-12; Fahey et al., 1983, In: Radioprotectors and anticarcinogens, pp. 103-120, Academic Press, New York). Another difficulty hampering the measurement of free cysteine during sample preparation is the presence of enzymes which degrade glutathione, such as y-glutamyltranspeptidase, to cysteine. Indeed, previous studies have suggested that the high cysteine levels observed in tissues such as kidney and liver are artifacts caused by y-glutamyltranspeptidase degradation of glutathione (Standeven and Wetterhahn, 1991, Toxicol. Appl. Pharmacol. 107:269-284).
There has been a long-felt but unfilled need for methods to diagnose tumors in mammals, particularly in the case of tumors which avoid diagnosis using conventional methods. There is also a need to elucidate and measure resistance of tumors to therapies, such as radiation and chemotherapy, and to adjust the therapy for treatment of such tumors. The present invention meets these needs.